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Official websites use. Share sensitive information only on official, secure websites. Competing Interests: The authors have declared that no competing interests exist. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In our study, core-shell nanoparticles containing lysozyme were formulated with precipitation and layering self-assembly. Factorial design DoE was applied by setting the process parameters during the preparation with Quality by Design QbD approach. The factors were the concentration of lysozyme and sodium alginate, and pH. Our aim was to understand the effect of process parameters through the determination of mathematical equations, based on which the optimization parameters can be predicted under different process parameters. Based on our results, we found that pH was the most important factor and pH 10 was recommended during the formulation. The mixing time had the best influence on the encapsulation efficiency and the particle size, which leads to the conclusion that a mixing time of 1 h is recommended. The novelty in our study is the presentation of a mathematical model with which the secondary structure of the protein and other optimization parameters can be controlled in the future during development of nanoparticle based on the process parameters. Recently, the use of proteins in the biomedical field has become more extensive, and they have been investigated intensively as potential biopharmaceutical drugs \[ 1 \]. They have well-known high specificity, complexity and low toxicity compared to small drug entities, but at the same time a number of barriers need to be overcome for the development of stably absorbable delivery systems \[ 2 \]. Lysozyme LYS is a harmless natural antimicrobial enzyme protein that can be derived from plants, animals and microorganisms as a single chain polypeptide. Hen-egg white is a common source in LYS separation studies, which is mainly performed by the precipitation method of egg-white proteins upon the addition of salts, solvents or the reduction of ionic strength \[ 5 \]. The diversity of the source renders LYS more affordable and cost-effective protein for the investigations \[ 6 \]. The antimicrobial activity of lysozyme is attributed to the destruction and lysation of the cell wall of Gram-positive bacteria and some fungi \[ 7 \]. Moreover, in vitro studies prove the activity of LYS against many Gram-negative bacteria including Pseudomonas aeruginosa \[ 8 \]. Pharmaceutically, nanotechnology has been performed to improve drug delivery performance, basically by improving bioavailability through the administration of the drug entity in nanoscale particles NPs or molecules in the range of nm which can overcome the biological barriers, targeting the absorption site, enhancing stability and solubility by increasing the surface area \[ 9 \]. Therefore, various nanotechnology-based drug formulations have been introduced to the market for treating and controlling numerous diseases, such as cancer, central nervous system diseases and infections \[ 10 \]. Moreover, the delivery of the drug in the form of NPs protects the natural products, such as proteins, from degrading enzymes, as well as controls the release of the incorporated bioactive molecules \[ 11 \]. Nanocarrier systems used extensively for protein delivery based on synthetic polymers, liposomes and metal have been replaced as a result of many limitations, e. Accordingly, protein NPs and their conjugates have replaced nanocarrier systems, offering the advantages of nanosized structure with good biopharmaceutical characteristics. Also, their production is cost-effective and easy to tailor to meet the specific requirements \[ 13 \]. The precipitation technique bottom-up approach represents the most applicable method for the production of NPs for both small-scale and bulk production, owing to its simplicity, low energy input, low generated temperature and cost-effectiveness compared to the other top-up methods \[ 14 \]. Zhang et al. Moreover, LYS complexed with different concentrations of sodium alginate showed two stages of aggregates with loss of activity based on the alginate content, but antimicrobial activity was recovered upon the addition of calcium chloride \[ 18 \]. The in vitro cytotoxicity of the complex was found to be dose-dependent \[ 19 \]. Polyelectrolyte multilayers are well-defined nanostructure with some potential applications, such as biomaterial coatings \[ 20 \]. Also, polyelectrolyte core-shells of bovine serum albumin nanoparticles BSA-NPs developed through the layer-by-layer LBL technique were used as a carrier system to control the release of ibuprofen. The inner layer was anionically made from poly sodiumstyrene sulphonate, and the cationic outer layer from CS, which enabled interaction with the negatively charged cell membrane and facilitated cell up-take \[ 21 \]. Most importantly, the technique is usually conducted under normal experimental conditions and mainly in an aqueous solution, hence it is suitable to encapsulate proteins and polypeptide drugs \[ 24 \], and recently the application of three layers of polyelectrolyte polymers in anticancer NPs demonstrated cancer-cell targeting with efficient internalization \[ 25 \]. The aim of our present work is to formulate, analyse and optimize core-shell type NPs containing LYS, as well as to write mathematical relationships between process parameters and product parameters. The preparation of LYS NPs was made according to 2 3 full factorial design; therefore 8 samples were prepared. Namely, 0. The 8 samples were centrifuged at rpm for 15 minutes by using a Hermle ZK high performance refrigerated centrifuge Hermle AG, Gossheim, Germany. Upon redispersion the total amount of precipitated LYS aqueous alginate solutions 25 ml of conc. The samples were mixed with a high shear mixer Ultra-Turrax, Germany for 15 seconds, followed by mixing for 1 h and 2 h. Following the guidelines of QbD, the steps shown in Fig 1 were performed \[ 27 \]. Based on the results of the risk analysis, the next step was to select the factors for the factorial experimental design. The factors selected were the concentration of alginate, pH and mixing time, which were used on 2 levels in DOE Table 1. Based on the absorbance, the concentration of unprecipitated NP enzymes was used to calculate precipitation efficiency. The obtained supernatants were carefully separated from the encapsulated NPs and absorption was measured at nm for each sample based on a pre-recorded calibration line. The concentration of free enzyme NPs was measured for all samples, from which encapsulation efficiency was determined. The Zeta potential of the same sample was measured with a Malvern Zetasizer apparatus with three parallel measurements Malvern Instruments, Malvern, UK. The structure and the morphology of the NPs after layering were described with transmission electron microscopy TEM. The samples were suspended in ethanol and dropped onto a carbon film-coated copper grid. The absorptions of the bacterial suspension were measured for 5 minutes before each test to reduce the error arising from bacterial sedimentation. LYS activity was calculated from the percentage degradation of the bacterial cells relative to crude LYS activity as a reference. The infrared spectra of the prepared samples and the other excipients were obtained with a FT-IR Avatar FT-IR ThermoScientific, USA apparatus, by using the potassium bromide disc method, scanning was run in the wavelength range of to cm -1 , the spectra were collected from 64 scans to obtain smooth spectra, at the spectral resolution of 4 cm -1 and applying CO 2 and H 2 O corrections. The SpectraGryph version 1. Friedrich Menges Software-Entwicklung, Germany software was used for the second derivation of spectra. For the deconvolution of the second derivatives, the Fityk software was used \[ 28 \]. After assigning the peaks, the area under the curve was calculated. For the measurements, a 4-opened quartz cuvette with an optical length of 1 cm was used, and the solid samples were dissolved in PBS buffer applying a protein concentration of 0. The spectra were corrected with the PBS buffer background. After the precipitation step, precipitation efficiency was calculated according to the UV spectra of the supernatant after centrifugation. In this case, average precipitation efficiency was The next step was the layering of alginate with alginate solution of different concentrations and different pH values. From these data, the loss of LYS was calculated and summarized with precipitation efficiency, after which encapsulation efficiency can be calculated. EE was between After the layering step, the concentration of LYS of the supernatant was very low after centrifugation. It can be explained by the electrostatic relationship between LYS and polyanionic alginate because the redispersion procedure was performed directly in the alginate solution and LYS could not solve in the buffer because the formation of the alginate layer on the surface of the precipitated LYS started immediately. The alginate layer formed can protect LYS. The effects of mixing time and pH were important factors, but statistically not significant. Therefore, the values of the coefficients were very low and statistically not significant. An inverse relationship can be seen between mixing time and EE Fig 2. Therefore, increasing mixing time is not recommended. During a mixing time of 1 h the alginate layer can be formed, which was confirmed by the negative Zeta potential values in all cases. Fig 2 reveals that this factor had an effect on EE only in the lower pH range. In the higher pH range dissolution did not start after a mixing time of 2 h. The third factor was alginate concentration, but this effect was very low 0. In this case, a low linear relationship was detected between the factor and EE. The predicted and the observed values can be seen in Fig 2. The predicted values correlate well with the observed values. This mathematical model can be used to show that EE can be predicted well in this range. Particle size was measured freshly before lyophilisation with the laser diffraction method. In each case, it can be seen that the final particle size was smaller than after the first step of preparation. The reason for this is that the polymer layer can result in a more compact NP structure. It can be seen in Fig 2 that mixing time had the greatest effect on particle size. During mixing, the dissolution of LYS can start from the NPs, and the degradation of the polymer can also start in parallel with this process. This can cause a decrease in particle size. In this case x 3 the coefficient was -9 Eq 5 , which means an inverse relationship between particle size and mixing time. The alginate concentration had a smaller effect on particle size. The coefficient was 5. It can be explained by the fact that a higher alginate concentration can result in higher layer thickness, which can lead to larger particle size. The coefficient of pH x 2 was 3. Fig 2 shows that here the predicted value also correlates well with the observed value, therefore this mathematical model is well applicable to predicting particle size in this range of parameter setting. The alginate layer on the surface of the precipitated LYS can be observed well Fig 3. The particle size correlated with the results determined with the Mastersizer based on the TEM, approximately particles around nm are visible. The core-shell structure is clearly visible in the TEM images, which is also supported by the Zeta potential values. Enzyme activity was measured according to the speed coefficient of the degradation of Micrococcus lysodeicticus cell wall. Table 4 shows the enzyme activity results for all samples prepared according to factorial design. Enzyme activity was between Based on the statistical evaluation, the effect of factors on enzyme activity can be seen on the response surface. If the pH is much lower than the IEP, the secondary structure of the protein may change. The following equation was obtained as the output of the statistical analysis:. In this case, only b 0 was a statistically significant factor, which means the average value. Alginate concentration x 1 had the largest effect on enzyme activity 7. In this range mixing time had no significant effect. The two-way interaction coefficients were also high for x 1 x 2 and x 1 x 3. The correlation between the predicted and the observed values can be seen in Fig 2. It can show the accuracy of the calculated mathematical model for enzyme activity. This means that enzyme activity can predict well in this range with the application of this mathematical model. The amide I region can be found between — cm -1 \[ 30 \]. After the second derivation of the — cm -1 region, the deconvolution of the peaks was performed, the results of which are shown in Fig 5. Seven main peaks were found in this region. This may be due to freeze-dried LYS because this product may be more sensitive to environmental parameters than spray-dried LYS. In these cases, both alginate concentration and pH were at minimum levels. However, Szigeti et al. This tendency correlates very well with the enzyme activity results Fig 2. The effects of all factors were positive Eq 7 , which means a linear relationship between the factors and the optimization parameter. Based on the CD measurements, the protein chain unfolds during the synthesis of LYS-based NPs, while the alginate shell causes a more compact structure because it wraps and compresses the chains of the precipitated protein. The reason for this may be that the FTIR measurement was performed in solid state of protein, while CD spectroscopy was measured in liquid. In this study, a simple procedure and analysis for the preparation of core-shell NPs containing LYS were presented. The secondary structure of all samples was determined and statistically evaluated. These optimization parameters correlate well each other. The coefficient of the effect of mixing time was the highest for encapsulation efficiency and particle size, since the dissolution of LYS started during mixing, therefore a mixing time of 1 h is recommended during formulation. In the case of alginate layered and raw material LYS, the difference was very high because of the liquid form during the CD measurements. Mathematical models were set up successfully in accordance with the QbD guidelines, which can be used to predict future optimization parameters and design space determination in this range. In summary, this information may help the design of the formulation in the future because it was a very simple composition with a minimal number of excipients applied, therefore only the factors can affect the optimization parameters no other effects should be considered. All relevant data are within the paper and its Supporting Information files. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. PLoS One. Find articles by Reihaneh Manteghi. Find articles by Ditta Ungor. Reihaneh Manteghi : Formal analysis, Investigation, Writing — original draft. Ditta Ungor : Formal analysis, Investigation. Abbas Farmany : Editor. Received Mar 30; Accepted Nov 13; Collection date Open in a new tab. Sample c alg. Click here for additional data file. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.

Buying powder Osterhout